Experimental vaccination by single dose sporozoite injection of blood-stage attenuated malaria parasites.

Malaria vaccination approaches using live Plasmodium parasites are currently explored, with either attenuated mosquito-derived sporozoites or attenuated blood-stage parasites. Both approaches would profit from the availability of attenuated and avirulent parasites with a reduced blood-stage multiplication rate. Here we screened gene-deletion mutants of the rodent parasite P. berghei and the human parasite P. falciparum for slow growth. Furthermore, we tested the P. berghei mutants for avirulence and resolving blood-stage infections, while preserving sporozoite formation and liver infection. Targeting 51 genes yielded 18 P. berghei gene-deletion mutants with several mutants causing mild infections. Infections with the two most attenuated mutants either by blood stages or by sporozoites were cleared by the immune response. Immunization of mice led to protection from disease after challenge with wild-type sporozoites. Two of six generated P. falciparum gene-deletion mutants showed a slow growth rate. Slow-growing, avirulent P. falciparum mutants will constitute valuable tools to inform on the induction of immune responses and will aid in developing new as well as safeguarding existing attenuated parasite vaccines.

A function of profilin in force generation during malaria parasite motility that is independent of actin binding.

During transmission of malaria-causing parasites from mosquito to mammal, Plasmodium sporozoites migrate at high speed within the skin to access the bloodstream and infect the liver. This unusual gliding motility is based on retrograde flow of membrane proteins and highly dynamic actin filaments that provide short tracks for a myosin motor. Using laser tweezers and parasite mutants, we previously suggested that actin filaments form macromolecular complexes with plasma membrane-spanning adhesins to generate force during migration. Mutations in the actin-binding region of profilin, a near ubiquitous actin-binding protein, revealed that loss of actin binding also correlates with loss of force production and motility. Here, we show that different mutations in profilin, that do not affect actin binding in vitro, still generate lower force during Plasmodium sporozoite migration. Lower force generation inversely correlates with increased retrograde flow suggesting that, like in mammalian cells, the slow down of flow to generate force is the key underlying principle governing Plasmodium gliding motility.

Malaria parasite LIMP protein regulates sporozoite gliding motility and infectivity in mosquito and mammalian hosts.

Gliding motility allows malaria parasites to migrate and invade tissues and cells in different hosts. It requires parasite surface proteins to provide attachment to host cells and extracellular matrices. Here, we identify the Plasmodium protein LIMP (the name refers to a gliding phenotype in the sporozoite arising from epitope tagging of the endogenous protein) as a key regulator for adhesion during gliding motility in the rodent malaria model P. berghei. Transcribed in gametocytes, LIMP is translated in the ookinete from maternal mRNA, and later in the sporozoite. The absence of LIMP reduces initial mosquito infection by 50%, impedes salivary gland invasion 10-fold, and causes a complete absence of liver invasion as mutants fail to attach to host cells. GFP tagging of LIMP caused a limping defect during movement with reduced speed and transient curvature changes of the parasite. LIMP is an essential motility and invasion factor necessary for malaria transmission.

A unique profilin-actin interface is important for malaria parasite motility.

Profilin is an actin monomer binding protein that provides ATP-actin for incorporation into actin filaments. In contrast to higher eukaryotic cells with their large filamentous actin structures, apicomplexan parasites typically contain only short and highly dynamic microfilaments. In apicomplexans, profilin appears to be the main monomer-sequestering protein. Compared to classical profilins, apicomplexan profilins contain an additional arm-like ß-hairpin motif, which we show here to be critically involved in actin binding. Through comparative analysis using two profilin mutants, we reveal this motif to be implicated in gliding motility of Plasmodium berghei sporozoites, the rapidly migrating forms of a rodent malaria parasite transmitted by mosquitoes. Force measurements on migrating sporozoites and molecular dynamics simulations indicate that the interaction between actin and profilin fine-tunes gliding motility. Our data suggest that evolutionary pressure to achieve efficient high-speed gliding has resulted in a unique profilin-actin interface in these parasites.

Coupling of Retrograde Flow to Force Production During Malaria Parasite Migration.

Migration of malaria parasites is powered by a myosin motor that moves actin filaments, which in turn link to adhesive proteins spanning the plasma membrane. The retrograde flow of these adhesins appears to be coupled to forward locomotion. However, the contact dynamics between the parasite and the substrate as well as the generation of forces are complex and their relation to retrograde flow is unclear. Using optical tweezers we found retrograde flow rates up to 15 µm/s contrasting with parasite average speeds of 1-2 µm/s. We found that a surface protein, TLP, functions in reducing retrograde flow for the buildup of adhesive force and that actin dynamics appear optimized for the generation of force but not for maximizing the speed of retrograde flow. These data uncover that TLP acts by modulating actin dynamics or actin filament organization and couples retrograde flow to force production in malaria parasites.